Purification and characterization of a membrane-associated cAMP-binding protein from developing Dictyostelium discoideum.

Cyclic AMP-binding protein and estrogen receptor: Antagonism during nuclear translocation in a hormone-dependent mammary tumor

Persistent cAMP binding by the chemotactic cAMP receptor in the presence of a detergent in Dictyostelium discoideum

Adenylate cyclase and cAMP-dependent protein kinase activities in gametocytogenic (LE5) and nongametocytogenic (T9/96) clones of Plasmodium falciparum were compared to explore the role of cAMP in sexual differentiation of the parasite. Basal adenylate cyclase levels were equivalent in the 2 clones. However, cAMP-dependent histone II-A kinase activity was significantly higher in LE5 than in T9/96 over a range of cAMP concentrations. This difference was due to a decreased Vmax for the enzyme in the nongametocytogenic clone and not to an increased Ka for cAMP. Examination of parasite cAMP-binding proteins, likely to be kinase regulatory subunits, by both photoaffinity labeling with [32P]8-N3-cAMP and affinity chromatography of metabolically [35S]methionine-labeled cytosol of cAMP-agarose revealed a 53-kDa cAMP binding protein in both clones and a 49-kDa cAMP-binding protein in T9/96 that was absent in LE5. Our results suggest that T9/96 has lost the ability to undergo gametocytogenesis due to a substantial decrease in cAMP-dependent protein kinase activity rendering the parasite unable to respond to increased intracellular cAMP levels. Moreover, the reduction in cAMP-dependent protein kinase activity may be due to the presence of an alternative regulatory subunit of the kinase.

The hormonal regulation of cAMP-dependent protein kinase was examined in granulosa cells from diethylstilbestrol-implanted immature rats. Follicle-stimulating hormone (FSH) increased the number of available cAMP-binding sites in a dose- and time-dependent manner, with a maximum 4-6-fold increase at 50-100 ng/ml between 6 and 48 h of culture after a transient decrease in available sites during the first 6 h. The potent gonadotropin-releasing hormone (GnRH) agonist [D - Ala6]des - Gly10 - GnRH - N - ethylamide (GnRHa) reduced the FSH-induced increase in cAMP-binding sites by approximately 50% at 24 and 48 h of culture. Photoaffinity labeling with 8-azido-[32P] cAMP revealed the existence of one major cAMP-binding protein (Mr = 55,000 +/- 400) which appeared to be the regulatory (R) subunit of type II cAMP-dependent protein kinase. While FSH induced a 5-10-fold increase in the labeling of R II both in vivo and in vitro, GnRHa reduced the amount of R II induced by FSH in granulosa cells cultured for 48 h. The large increase in R II subunit was not accompanied by a corresponding increase in protein kinase activity, which was only enhanced by 50% after 48 h of culture with FSH. Fractionation of granulosa cell cytosol from FSH-treated ovaries on DEAE-cellulose showed a single peak of cAMP-dependent phosphokinase activity with the elution properties of a type II protein kinase. However, the peak of cAMP binding activity (eluted at 0.20 M KCl) was not coincident with the protein kinase activity. FSH transiently stimulated cAMP-dependent protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early increase in protein kinase activity, causing a delay in activation until 60 min. These findings suggest that a large dose- and time-dependent increase in the content of cAMP-binding sites may be a major factor in cAMP-mediated differentiation of granulosa cells. The inhibitory effect of GnRHa on both FSH-induced protein kinase activation during the first minutes of culture and on FSH-induced R II synthesis during the subsequent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agonists.

When cells of Dictyostelium discoideum are starved they aggregate to form multicellular organisms that eventually can differentiate into fruiting bodies of spores and stalk cells, cAMP plays a dual role in this process: it is the chemotactic agent that allows cells on a moist surface to aggregate to a common point to form the multicellular organism [1], and it is also involved in the process of differentiation. Pulsatile additions of cAMP stimulate cell differentiation [2,3]. High concentrations of cAMP can induce stalk cell formation [4,5] and, under certain conditions, spore cell production can be induced by cAMP [6,7]. Cyclic AMP has also been shown to induce differentiation in the multicellular aggregate [8]. Further evidence of the involvement of cAMP in differentiation is given by the finding that addition of cAMP to cells affects the expression of developmentally regulated proteins [9] and the expression of specific genes [10]. Receptors for cAMP have been identified by many workers. Membrane-associated cAMP-binding proteins have been reported [11-14], and soluble cAMP-binding proteins have also been identified [14-19]. There has been a report that the soluble cAMP-binding proteins are associated with protein kinase activity [16] but this report has not been confirmed [17,18]. Wallace and Frazier [14] reported that the soluble cAMP-binding proteins are of M r 26 000, 33 000, and 36 000, but Cooper et al. [19] have reported that the M r are higher, in the 36 000-40 000 range. Here we describe our studies indicating that the endogenous cAMP-binding protein of M r 40 000 is extremely sensitive to endogenous protease activity, and the lower M r binding proteins are produced during the process of freezing the cells. When fresh extracts are used, only the 40 000 M r binding protein is observed. Tos-LysCH2C1, an inhibitor of proteinase I of Dictyostelium [20,21] inhibits this degradative reaction.

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.

Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP- dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP- binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP- dependent kinases.

Increasing the flexibility of the LANCE cAMP detection kit.

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from [formula omitted] plasma membranes

PROTEIN KINASE ACTION AS A MEDIATOR OF CHEMOTAXIS AND DIFFERENTIATION IN THE CELLULAR SLIME MOLD, DICTYOSTELIUM DISCOIDEUM

Three binding proteins, one specific for guanosine 3′: 5′-monophosphate (cGMP) and two specific for adenosine 3′: 5′-monophosphate (CAMP) have been partially purified from vegetative cells of Myxococcus xanthus M300. The cGMP binding activity was found in the periplasmic shock fluid. Scatchard analysis indicated only a single class of binding sites with high affinity (apparent KD, 42 nM). The two cAMP binding activities were physically distinct, as indicated by their elution patterns from DEAE-cellulose, KD values and cellular locations. The cytoplasmic cAMP binding protein, which is probably identical to that previously isolated from developing myxospores of M. xanthus had an apparent KD of 57 nM, whereas the periplasmic cAMP binding protein had an apparent KD of 1 PM. During development, the nucleotide binding proteins exhibited changes in activities consistent with their postulated roles during fruiting body development.

The small GTPase Rap1 has been implicated in a variety of cellular processes including the control of cell morphology, proliferation, and differentiation. Stimulation of a large variety of cell surface receptors results in the rapid activation of Rap1, i.e. an increase in the GTP-bound form. This activation is mediated by second messengers like calcium, cAMP, and diacylglycerol, but additional pathways may exist as well. Here we describe a ubiquitously expressed guanine nucleotide exchange factor of 200 kDa that activates Rap1 both in vivo and in vitro. This exchange factor has two putative regulatory domains: a domain with an amino acid sequence related to cAMP-binding domains and a PDZ domain. Therefore, we named it PDZ-GEF1. PDZ-GEFs are closely related to Epacs, Rap-specific exchange factors with a genuine cAMP binding site, that are directly regulated by cAMP. The domain related to cAMP-binding domains, like the cAMP binding site in Epac, serves as a negative regulatory domain. However, PDZ-GEF1 does not interact with cAMP or cGMP. Interestingly, PDZ-GEF1 also activates Rap2, a close relative of Rap1. This is the first example of an exchange factor acting on Rap2. We conclude that PDZ-GEF1 is a guanine nucleotide exchange factor, specific for Rap1 and Rap2, that is controlled by a negative regulatory domain.

A cAMP-binding protein and cAMP-dependent protein kinase have been identified and partially characterized in bovine adrenocortical plasma membranes. [3H]cAMP binding to the plasma membrane preparation demonstrates both high (Ka = 4.2 X 10(9) M-1) and low (Ka = 1.3 X 10(8) M-1) affinity binding sites. cAMP-dependent protein kinase was demonstrated using both bovine histone and endogenous substrates. The detergent-dependent solubilization characteristics of this cAMP-dependent protein kinase indicate that it is an integral membrane protein.

Although cAMP binding to wild type cAMP receptor protein (CRP) induces specific DNA binding and activates transcription, cyclic nucleoside monophosphate (cNMP) binding to the CRP mutant Ser128 --> Ala does not, whereas the double CRP mutant Thr127 --> Leu/Ser128 --> Ala activates transcription even in the absence of cNMP. Isothermal titration calorimetry measurements on the cNMP binding reactions to the S128A and T127L/S128A mutants show that the reactions are mainly entropically driven as is cAMP binding to CRP. In contrast to cAMP binding to CRP, the binding reactions are noncooperative and exothermic with binding enthalpies (DeltaHb) ranging from -23.4 +/- 0.9 kJ mol-1 for cAMP binding to S128A at 39 degrees C to -4.1 +/- 0.6 kJ mol-1 for cAMP binding to T127L/S128A at 24 degrees C and exhibit enthalpy-entropy compensation. To account for the inactivity of the S128A mutant, in vitro and in vivo DNA binding experiments were performed on the cAMP-ligated S128A mutant. The cAMP-ligated S128A mutant binds to the consensus DNA binding site with approximately the same affinity as that of cAMP-ligated CRP but forms a different type of complex, which may account for loss of transcriptional activity by the mutant. Energy minimization computations on the cAMP-ligated S128A mutant show that amino acid conformational differences between S128A and CRP occur at Ser179, Glu181, and Thr182 in the center of the DNA binding site, implying that these conformational changes may account for the difference in DNA binding.

Evidence that a cAMP binding protein from Dictyostelium discoideum carries S-adenosyl- l-homocysteine hydrolase activity